Several weeks back I posted a question of how to map a new gene (who has RFLP
inbreds etc). Thanks to all who responded. Many people were very interested
in this question and just wrote to ask for the answers, so here are all of the
the responses I got with answers.
Mark Guiltinan
Penn State Biotechnology Institute
mjg at psupen.psu.edu
Message for Mark Guiltinan
From: GLAZEBROOK at Frodo.MGH.Harvard.EDU
Date: Fri, Nov 5, 1993 10:39 AM
Subject: RE: How to Map a New Gene
To: Mark Guiltinan
Dear Mark,
For cloned genes, the easiest way to get a really good map position is use the
recombinant inbred lines from Caroline Dean's lab. They were just described as
a technical advance in the latest Plant Journal. I'm not sure if any of the
staock centers have them, or if you have to get them from the Dean lab. You
have to find a col/La-er polymorphism in your gene, and then either make it
into a CAPS marker (see Konieczny and Ausubel, plant journal issue one before
the current one) or use it as an RFLP, then type the recombinant inbred lines
for your gene. By comparing to the known map data for the recombinant inbred
lines, you can work out where your gene is.
Jane Glazebrook
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Message for Mark Guiltinan
From: PLZMLH at VAX.CCC.NOTTINGHAM.AC.UK
Date: Thu, Nov 4, 1993 9:39 AM
Subject: RE: How to Map a New Gene
To: Mark Guiltinan
Dear Mark,
If you have a molecular probe for your gene (and it sounds
as though you have) then you can use the recombinant inbred lines
produced by Caroline Dean and Clare Lister (Columbia and Landsberg)
or Pablo Scolnik (WS X W100). Which backgound is your mutant in?
This will dicate which set to use.
Both ABRC and we supply the seed for these lines. For practiacl help
with the mapping I suggest you contact Caroline Dean or Pablo
Scolnik. They have the mapping data for the respective lines.
If you need anymore information, please do not hesitate to
contact me.
Mary Anderson
Director of the Nottingham Arabidopsis Stock Centre
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Message for Mark Guiltinan
From: DELANEYT 919-541-8577
Date: Tue, Nov 9, 1993 7:58 AM
Subject: RE: How to Map a New Gene
To: Mark Guiltinan
Dear Mark,
You need to do several things:
1. using your cloned sequence as a probe, test a bank of Southern blots with
the A.t. ecotypes Col-O, La-o and Nd-O, cut with a variety of enzymes (e.g.
BamHI, BglII, EcoRI, HindIII, PstI for starters). Look for an enzyme, that
your probe displays a polymorphism
(band shift, or band cleavage) in any two or more ecotypes. When you find such
an enzyme/ecotype combination, go to 2:
2. Contact either Dr. Elliot Meyerowitz' lab or Dr. Howard Goodman's lab
(Calthech, Mass Gen'l, resp), to ask for their assistance; -they have banks of
Southern blots, that may throw your probe onto. You provide them with the
enzyme that you identified in "1", that shows an
RFLP in Col-O versus La-O, Col-O vs Nd-O, or La-O vs Nd-O. Realize that Dr.
Goodman's filters do not include Nd-O, so if you require this ecotype to see a
polymorphism, you will need to contact Dr. Meyerowitz' lab.
3. They will often help people out. In that case, you would send them your
probe, the nature of the RFLP, and someone in their lab wuoild do the hyb and
analysis, providing you with a map position.
You could do the same thing by making you own DNA from the recombinant inbred
line that are available, but it isn't reasonable to do unless you have _many_
probes to map. You could
alternatively map your RFLP (from "1") versus the wide assortmant of published
RFLPs, RAPDs, SSLPs and phenotypic markers, thus mapping your clone to those
markers; -but the efficient and economical (and faster) approach is above
(1-3).
A similar stategy would apply with RAPDs or SSLPs, but I'm not aware of that
service being provided to the community, as is true for RFLPs (we should all be
grateful for the services provided by the aforementioned labs!).
Hope this helps,
Terry Delaney
Ciba Agric Biotech
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